A crucial element in the development of effective therapeutic and prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. The SIV infection of macaques is such a model since this virus induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Therefore, candidate vaccines can be evaluated not only for their ability to prevent infection but also for their ability to prevent AIDS. The major vaccine effort within the laboratory has been an evaluation of the highly attenuated modified vaccinia virus Ankara (MVA) strain as a recombinant vector used for priming that is followed by a subunit SIV boost. Interest in this approach is based upon a pilot study of 12 macaques in which animals immunized with the MVA-SIV recombinant exhibited significant down modulation of virus replication that was associated with prolonged survival. Two of these vaccinated macaques have remained healthy for 4 years. Modulation of viremia appeared to correlate with a gag-specific antibody response observed prior to SIV challenge. These studies demonstrated that MVA was as effective as conventional vaccinia viruses such as the New York Board of Health in eliciting an immune response in monkeys. In addition, this is the first study to demonstrate that immunization can modulate viremia and disease progression in the absence of sterilizing immunity in the SIV/macaque model for AIDS.We wished to explore MVA as a live virus vaccine in the SIV model with the eventual goal of optimizing this approach for future development as a potential AIDS vaccine for humans. The short-term goals of this project are twofold: to define the antigens required to mediate protection and to investigate the role of gag-specific CTL in this effect. Three second generation recombinant MVA viruses were generated that expressed either gag-pol alone, env alone, or a combination of gag-pol and env, with all genes being expressed from the strong early/late synthetic promoter. A cohort of 24 rhesus macaques have been immunized (six per immunogen) to evaluate immunogenicity with the eventual goal of analyzing the kinetics of viremia in these animals after intravenous challenge and attempt to correlate any effects on viral load with immunogen. Four sequential immunizations resulted in moderate boosting of both gag and env-specific antibody responses. None of the animals developed neutralizing antibody specific for the challenge strain. These macaques were challenged with cell free SIVsmE660 and all of the macaques became infected. However, plasma viremia in each of the groups immunized with MVA-SIV recombinants had significantly lower viral load (p=0.03) than the group vaccinated with no recombinant MVA. There was no significant difference in viral load between the groups immunized with MVA-SIV recombinants. Seven vaccines have maintained low viremia and normal CD4 numbers; whereas, all of the control macaques have developed CD4 depletion. These data demonstrate that vaccination with MVA-SIV recombinants results in significant protection from high viremia and AIDS presumably mediated through cell mediated immunity.Evaluation of gag-specific CTL in vaccinia-immunized macaques presents a considerable challenge; and therefore, in collaboration with Dr. Norman Letvin, utilized macaques expressing the MamuA*01 MHC Class I haplotype for which a dominant gag-specific epitope (p11 C) has been identified. In order to select MamuA*01 macaques, we developed PCR primers, and conditions to screen 68 macaques and identified six animals which expressed this haplotype. Four of these macaques were immunized with the MVA-gag-pol recombinant virus at 0 and 13 weeks and an additional two macaques were immunized with MVA and sequential generation of a p11C-specific CTL response was measured utilizing both traditional CTL assays as well as MHC Class I/Peptide tretramer staining. Macaques immunized with MVA expressing gag-pol developed CD8+ CTL as early as 2 weeks after the first immunization (2 of 4) which was boosted in all animals to high levels after the second immunization. CTL could be demonstrated in 1 to 3% of peripheral blood lymphocytes and lymph nodes by MHC Class I/Peptide tretramer staining after the second immunization. These levels are equivalent to those observed in SIV-infected macaques suggesting that this vaccine strategy approaches the cell mediated immune response achieved in such approaches as attenuated live virus vaccines. Challenge of these macaques with SIVsmE660 resulted in a rapid and substantial anamnestic CTL response. Viral load set-point was reduced in the macaques immunized with MVA-Gag-pol as compared to controls. However, the difference was not statistically significant. Nevertheless, much of the spread in viremia in the immunized macaques could be explained by their response to immunization since there was an inverse correlation between the magnitude of the CTL response during vaccination and plasma viral load set-point established after SIV challenge.